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Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB.

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Zuschriften
DOI: 10.1002/ange.200801184
Enzyme Mechanisms
Cage Escape Competes with Geminate Recombination during Alkane
Hydroxylation by the Diiron Oxygenase AlkB**
Rachel N. Austin,* Kate Luddy, Karla Erickson, Marilla Pender-Cudlip, Erin Bertrand,
Dayi Deng, Ryan S. Buzdygon, Jan B. van Beilen, and John T. Groves*
The alkane hydroxylase AlkB of Pseudomonas putida GPo1 is
typical of a large class of membrane-spanning diiron oxygenases that catalyze hydroxylation, epoxidation, and desaturation reactions.[1, 2] These enzymes are of considerable interest
due to their impact on global hydrocarbon metabolism,[3] their
potential for practical biocatalytic application, and the
resulting inspiration for the design of synthetic biomimetic
catalysts.[4] Although the three-dimensional structures of
AlkB or any closely related proteins are unknown, topology
modeling has predicted a structure comprised of six membrane-spanning helices with the catalytic iron diad appended
to the cytoplasmic termini of the helix bundle.[5] M(ssbauer
data and alanine scanning have suggested that the diiron
binding site is histidine-rich,[6] as found in hemerythrin, and in
contrast to the predominantly carboxylate binding motifs
found in the diiron hydroxylases sMMO[7] and T4 MOh.[8]
Through protein side-chain mutations, a long, hydrophobic
substrate-binding channel within the bundle has been identified that is tuned to accept medium-length alkanes.[5] AlkB
was the first alkane hydroxylase shown to generate a longlived substrate carbon radical during catalysis, as revealed by
diagnostic skeletal rearrangements of the hydrocarbon probe
norcarane.[9]
Herein we report results for the AlkB hydroxylation
reaction using a panel of radical-clock substrates that display
intrinsic rearrangement rates spanning five orders of magnitude, from a moderately slow 2.8 0 107 s 1 for bicyclo[3.1.0]hexane[11] to an ultrafast 1011 s 1 for trans-1-methyl-2-
[*] D. Deng, R. S. Buzdygon, Prof. J. T. Groves
Department of Chemistry, Princeton University
Princeton NJ 08544 (USA)
Fax: (+ 1) 609-258-0348
E-mail: raustin@bates.edu
Prof. R. N. Austin, K. Luddy, K. Erickson, M. Pender-Cudlip,
E. Bertrand
Department of Chemistry, Bates College
Lewiston ME 04240 (USA)
Fax: (+ 1) 207-786-8336
E-mail: jtgroves@princeton.edu
Dr. J. B. van Beilen
Department of Plant Molecular Biology, University of Lausanne
(Switzerland)
[**] We thank FMC, Princeton, for access to GC-MS instrumentation and
NSF (CHE-0221978 (R.N.A., J.T.G.) and CHE 0616633 (J.T.G.)), NIH
(GM 072506 (R.N.A.) and 2R37M036298 (J.T.G.)), the Henry
Dreyfus Foundation (R.N.A.), and the Howard Hughes Foundation
(R.N.A.) for support of this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801184.
5310
phenylcyclopropane.[12] Significantly, the ratios of rearranged
and unrearranged products (R/U) found for the three mostslowly rearranging substrates were all in the range of unity
even though their rearrangement rates differed widely. To
account for these unusual results we propose a new diffusional model of AlkB hydroxylation that involves radical
cagelike active-site dynamics of the type observed for hemeand cobalamin-containing metalloproteins.
AlkB from P. putida GPo1 was expressed in P. putida
GPo12 in the manner we have previously described.[13] GPo12
is a receptacle clone that has been stripped of its innate
hydroxylases and dehydrogenases. This approach has the
advantages of producing unambiguous protein expression and
high activity for only the inserted hydroxylase gene, while
showing otherwise negligible background oxidation. Substrates were oxidized in resting whole cells and in cell-free
extracts because all attempts to isolate and purify AlkB to
date have led to loss of activity. Results for the AlkBmediated oxygenation of the three alkane substrates, bicyclo[4.1.0]heptane (norcarane, 1), bicyclo[3.1.0]hexane (2), and
bicyclo[2.1.0]pentane (3), are presented in Table 1. These
simple alkanes were chosen because of their similar size,
nearly spherical shape, highly analogous structures, and
similar rearrangement chemistry (Scheme 1). The data for
all three substrates showed large amounts of rearrangement
products (> 50 %) consistent with the involvement of discreet
radical intermediates during the hydroxylation process.
Further, the ratios of primary to secondary alcohols formed
from norcarane and bicyclohexane are similar to the partition
ratios observed for bona fide radical reactions for these
substrates (ca. 2 and 10 %, respectively).
It is striking, however, that the ratios of rearranged
products to unrearranged products (R/U) for these three
substrates do not correlate with the 100-fold change in the
radical rearrangement rate constants for bicyclo[2.1.0]pent-2yl (kr = 2 0 109 s 1),[14] 2-norcaranyl (kr = 2 0 108 s 1), and
bicyclo[3.1.0]hex-2-yl (kr = 2.8 0 107 s 1).[11] We found the
average R/U values for the three substrates remarkably
constant (1.6, 1.6, and 4.7), corresponding to apparent radical
lifetimes of 0.78, 7.8, and 170 ns, respectively. Indeed,
bicyclohexane, with the slowest rearrangement rate, displayed
the most rearranged product and by far the longest radical
lifetime. The same effect was observed when norcarane and
bicyclohexane were oxidized as a mixture. By contrast, the
ultrafast rearranging probe trans-1-methyl-2-phenylcyclopropane (kr = 1011 s 1) was confirmed to afford only rearranged
products.[9, 15] Clearly, there is a discrepancy here between the
observed results for the more-slowly rearranging substrates
and expectations based on Arrhenius-type kinetic behavior
2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2008, 120, 5310 ?5312
Angewandte
Chemie
Table 1: Product distribution for the AlkB oxygenation of bicycloalkanes
1?3.
R/U
GPo1
whole
cell
0.5
(0.3)
14.1
(2.8)
13.8 (1.7)
[0.4
(0.2)][a]
GPo1
cell-free
0.4
(0.2)
29.9
(3.1)
13.3 (0.6)
[1.0
(0.3)][a]
exo: 8.9 (0.3)
1.0
endo: 54.7
(2.1)[b]
[8.2 (2.4)][a]
exo: 8.4 (0.4)
2.1 (1.6)[f ]
endo: 40.9 (0.6)
[1.9 (0.6)][a]
GPo1
whole cell 8.9
(0.8)
exo: 6.6(0.6)
52.5 (2.1) endo: 8.0(0.9)
GPo1
cell-free
exo: 4.9(0.4)
67.3 (0.8) endo: 8.9(1.0)
5.6
(0.4)
GPo1 whole
cell
GPo1 cell-free
4.2
22.1 (0.8)
[1.9 (0.6)][a]
Scheme 2. Geminate recombination and cage escape.
13.5 (0.8)
[nd][c]
5.3 (4.7)[f ]
47.3 (2.6)
34.6 (4.7)
18.1 (6.8)
1.9
54.7 (5.7)
45.3 (5.7)
nd[c]
1.2 (1.6)[f ]
[a] Yield of corresponding ketone in square brackets. [b] Standard error in
parentheses. Endo-3- and exo-2-norcaranol were difficult to resolve. MS
fragmentation indicated that endo-3-norcaranol was the predominant
isomer in every case. [c] Product not detected. [d] Ketone not detected.
[e] Mixture of 2- and 3-cyclopentenone. [f] Average R/U in parentheses.
Scheme 1. Radical rearrangements of bicycloalkanes 1?3. Enz-FeO:
diiron oxygenase AlkB.
for rearrangements of freely diffusing radicals. The structural
similarity of the three substrates makes it unlikely that some
bulk-medium effect is the cause of this timing compression.
However, the results can be accommodated by a mechanism
in which there is a step in the mechanism that occurs after
hydrogen abstraction from the substrate and before rearrangement of the intermediate radical.
Angew. Chem. 2008, 120, 5310 ?5312
We suggest a scenario in which hydrogen atom abstraction
of the substrate cyclopropyl-carbinyl C H leads to a caged
radical pair [Fe2O H CR] (RP; Scheme 2). Kinetic simulations
show (see Figure S4 in the Supporting Information) that
differences in radical rearrangement rates are masked in the
product analysis for such a reaction scheme if diffusive cage
escape to a solvent-separated radical pair (RPss) were to occur
at a rate (ke) that is similar to the rebound rate (kR) leading to
the product alcohol. Indeed, with ke kR = 1010 s 1, which are
very reasonable rates for such processes, the ratio R/U for
substrate rearrangements occurring slower than 1010 s 1
would all reflect the ?caging efficiency? ke/kR and would not
be much affected by the differences in kr, in accord with the
experimental observations. The result is analogous to the
commonly observed suppression of kinetic hydrogen isotope
effects by strong binding of the substrate. The nearly
complete rearrangement of trans-1-methyl-2-phenylcyclopropane in such a situation could reflect rapid rearrangement
within the initial radical cage (kr > kR), unusually fast cage
escape for this substrate (ke > kR), or both. The long, hydrophobic substrate channel in AlkB, similar to that found by Xray structure analysis for ToMOH,[16] could provide a pathway
for separation of the insipient substrate radical while assuring
its eventual return to the rebound intermediate Fe2O H.
Competitive rebound and cage escape also offers an explanation of the large substrate concentration effect on apparent
2-norcaranyl radical lifetime for AlkB.[10] Here, population of
the substrate channel with additional substrate molecules
could displace and reorganize active-site water and create a
logjam that reduces ke and reinforces reaction of the initial
radical pair (RP).
We favor this explanation over several, less likely alternatives. The hypothesis that the intramolecular rate constants
measured outside the enzyme might be drastically changed in
the enzyme is inconsistent with the unchanged rearrangement
branching ratios that are observed. The possibility that there
are two paths?a concerted process and a radical pathway?
appears unlikely given that the fastest-rearranging probe is
fully rearranged. Furthermore, that hypothesis would require
a fortuitous and unlikely change in the relative flux through
the two pathways to balance the changing rearrangement
rates.
There is abundant photophysical evidence for other
proteins and in small-molecule systems for competitive
2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.de
5311
Zuschriften
cage-escape and recombination events. A particularly analogous example is the adenosyl radical recombination with
cobalt(II)?cobalamin described in considerable detail by
Sension et al.[17, 18] In this case, the transient kinetics in water
indicate that the rate constants for in-cage radical recombination and cage escape are both approximately 109 s 1. While
there are many dynamic processes at the AlkB active site that
could affect cage escape, the intervention of a nearby water
molecule by the formation of a hydrogen bond as in [Fe2O
HиииOH2 CR] (RPss) is particularly likely. The intercalation of
solvent molecules between organometallic radical pairs has
been directly observed to occur in the picosecond time
regime.[19, 20] Rapid reorganization of active-site water is also
thought to be involved in the complicated diffusional kinetics
that occur after photodissociation of heme protein axial
ligands.[21, 22] For photodissociated [heme-FeII NO] and [hemeFeII O2] in myoglobin, ligand recombination and cage escape
are competitive.[23] Thus, even though there is almost no
enthalpic barrier to ligand recombination, there is an entropic
driving force that favors ligand diffusion into adjacent protein
cavities where NO, for example, is observed to persist for up
to a nanosecond.
We note that the curious stereochemical outcomes for
hydroxylation of chiral ethane and n-octane by sMMO[24] and
AlkB,[25] which both show 60 % racemization despite showing
very different radical lifetimes with other probes, could also
be explained by competitive cage recombination, water
reorganization, and radical escape, as well as structural
heterogeneity in the solvation state of the reactive diiron
intermediate. Further, pMMO may be an AlkB-like diiron
hydroxylase, increasing the potential relevance of mechanistic
work on diiron non-heme alkane-oxidizing enzymes.[26] Likewise, the partial allylic scrambling observed for cyclohexene
hydroxylation by cytochrome P450 and analogous behavior of
numerous other substrates are also potentially explained by
such processes.[27] Stochastic behavior of the incipient radical
and nearby water can lead to different trajectories and
reaction outcomes for different individual molecules as the
result of processes such as cage escape and water reorganization that contribute to the events but are not timed by the
rearrangement clock. Accordingly, while diagnostic rearrangements at enzyme active sites such as AlkB provide
clear information regarding the radical nature of substratebased intermediates, these processes should not be expected
to follow a strict molecular horology.
Experimental Section
AlkB GPo1 was transferred to P. putida GPo12 according to
procedures previously reported.[5] In the resting cell approach, cells
were centrifuged at OD = 1 and resuspended in 50 mm phosphate
buffer (pH 7.2). The substrate was provided by vapor transfer and the
cells incubated for 3?4 h as we have described.[13] After centrifugation, the supernate was extracted three times with ethyl acetate,
concentrated, and assayed by GC-MS. Cell-free extracts were
prepared from cell pellets that were resuspended in potassium
phosphate buffer containing 5 % glycerol (pH 7.4), sonicated, and
centrifuged. Reaction mixtures consisting of 1 mL of the cell broth,
1 mL of dithiothreitol (DTT) solution, 2 mL of the substrate and 33 mL
of a 0.36 m NADH solution were incubated for 2?3 h.[11] The reaction
5312
www.angewandte.de
was quenched by adding CH2Cl2 (1 mL), and then vortexed and
centrifuged. The CH2Cl2 layer was removed, dried with anhydrous
sodium sulfate, and analyzed by GC-MS.
Received: March 11, 2008
Published online: June 2, 2008
.
Keywords: alkanes и cytochrome AlkB и oxygenases и
radical clocks и reaction mechanisms
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2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2008, 120, 5310 ?5312
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